The present invention relates to a diagnosis reagent for parasitoses and allergies, to a method for the preparation of this reagent, and to a method of diagnosis of parasitoses and of allergies, notably worm parasitoses which causes an increase in the circulating specific immunoglobulins, such as IgE, IgG.sub.4 IgM, by means of this reagent.
Protection of man and of domestic animals against parasitic diseases represents one of the great world problems in the field of public health. If the developed countries have resolved a certain number of their parasitological problems, without, however, having entirely eliminated them, parasitoses still constitutes a major obstacle to the economic development of many countries, and in particular of countries in the hot regions of the globe, which are those which must reckon with the greatest human proliferation.
It is essential, in order to undertake an effective fight against the scourge represented by parasitoses, to be able to carry out a positive, reliable and early diagnosis of parasitoses.
If clinical examination, X-ray examination and information drawn from case histories are of great assistance for establishing a diagnosis, it is essentially due to biological methods and notably immunoparasitological methods that investigation techniques have profoundly developed.
It is at present frequent to refer to a parasitic etiology, a febrile manifestation scarcely judged as "hepatic", "urinary", or "allergic".
Numerous serological techniques have been thus proposed: cutaneous tests by intradermoreaction, agglutination tests of latex particles and of sensitized red blood corpuscles, hemoagglutination tests, precipitation, reactions on gelose, immunoelectrophoretic analyses, tests by immunofluorescence, complement fixation reactions, etc. . . . These up till now conventional diagnosis methods have been able to develop recently, since perfectly known and individualized antigen fractions have been available, and since also, the preservation and stabilization of these antigens has been mastered, as well as their standardization. All these methods have their advantages and their drawbacks; none is infallible. It is thus that A. CAPRON and Coll. (Path. Biol. 1970, Vol. 18, pages 357-365) compare the results obtained for immunological diagnosis of human echinococcosis by the four following methods:
(1) immunoelectrophoresis (I. E.),
(2) hemagglutination (H.M.G.),
(3) agglutination reaction on latex (A.L.), and
(4) the complement fixation reaction (C.F.R.).
The over-all results of the immunological diagnosis of hydatidosis obtained by these authors, are summarized in Table I below.
TABLE I __________________________________________________________________________ Number of Number of Percentage Observed patients patients of significant Method studied positive positivity minimum Maximum Average __________________________________________________________________________ I.E. 350 321 91.7% 1 18 4.8 R.F.C. 318 227 71.4% 1/2 1/256 1/20 H.M.G. 330 260 78.8% 1/512 1/512000 1/30000 A.L. 279 220 78.8% 1/2 1/512 1/50 __________________________________________________________________________
This Table establishes the percentages of positivity, as well as the lower and upper limits obtained for the various methods used. It is noted that immunoelectrophoresis has enabled 91.7% of cases to be detected. In all cases where the existence of a hydatidosis has been positively concluded, the latter has been confirmed by the operational route, and it has never been indicated that an immunological diagnosis is unnecessary. On the other hand A. CAPRON and Collab., have noted that a certain number of patients in whom the immunological response was negative, were indeed hydatidosis carriers. The set-back percentage was 15.4% for I.E., 29% for R.F.C., 21.4% for H.M.G. and 24% for A.L. Another immunodiagnosis method proposed, dealt with the radioallergosorption test or RAST. This test is based on the fact that the patients afflicted with parasitosis have abnormally high specific immunoglobulin E (IgE) levels. The method was described in detail by CESKA and Collab. (J. Allergy Clin. Immunol. 1972, 49, 1). Thus D. VERVLOET and Collab. (Rev. franc. Allergol. 1976, 16 (no. 2), pages 73-78) have used this RAST technique in 60 patients having surgically confirmed hydatidosis and have been able to establish 54 times, namely in 90% of the cases, the presence of IgE's specific with respect to soluble antigens of the hydatic cyst. They have also established a good correlation between the specific IgE levels measured by the RAST and the total IgE level. However, on the other hand, they have not found a correlation between the levels of the IgE antibodies and the level of the antibodies measured by the conventional immunoelectrophoresis techniques of complement fixation, immunofluorescence and hemagglutination. These authors, like also, for example Niklaus WEISS and Collab. ("The Lancet", (Dec. 2, 1978) who have used the RAST in patients infected with Schistosoma heamatobium and S. mansoni, or DESSAINT and Collab. (Immunol. 1975, 29, 813) who have measured the specific IgE's in subjects afflicted with hydatidosis and with bilharziosis, conclude that the RAST can be added to the "conventional battery" of immunological tests, without supplanting them, by reason of its insufficient reliability and due to the fact that it is not a quantitative test, but only qualitative. The histamine determination tests more reliable than the RAST should also be mentioned.
In another field, which is that of allergology, the present inventors, based on the work of SHELLEY (notably in NATURE, 1962, 195, p. 1181-1183; BLOOD, 1962, 19, p. 208-216; Trans. Stud. Coll. Physns Philad., 1964, 32, p. 15-19), taken up subsequently by HIRSCH and ZASTROW (J. Allergy Clin. Immunol. 1972, 50, p. 338-347) and SOIFER and HIRSCH (J. Allergy Clin. Immunol., 1975, 56, p. 127-132) have applied the degranulation test of human basophile granulocytes with a diagnosis accuracy of the order of 94%, using a leucocyte suspension enriched 10 to 20 times in basophilic granulocytes (with respect to the circulating blood), whose cells are fixed on a slide and dyed with toluidine blue (cf. F. LEYNADIER, H. LUCE and J. DRY, Rev. Franc. d'Allergol., 1977, 17 (no. 4), p. 215-218).
Starting from the knowledge of the fact that the production of considerable amounts of circulating specific IgE's is one of the characteristics of the immune response to the majority of worm parasitoses, the present inventors have sought to adapt the method of measuring IgE's by specific antigens by using suspensions enriched in IgE-carrying basophil granulocytes, placed on slides, which they had previously developed in the domain of allergology, to the diagnosis of various worm parasitoses.
Consequently it is an object of the present invention to provide a diagnosis reagent for parasitoses and allergies, and notably for worm parasitoses, which caused an increase in the circulating specific immunoglobulins fixed on the basophils, such as the IgE's in particular, which reagent enables extremely reliable diagnoses to be obtained and is relatively simple in use, whilst the techniques which enable it to be obtained ensure sufficient concentrations of basophils in the reagent for the results obtained in the presence of antigen, in the course of the diagnosis, to be statistically interpretable with full safety.
Accordingly, the present invention provides a diagnosis reagent for parasitoses and allergies, and notably for worm parasitoses, which cause an increase in the circulating specific immunoglobulins and notably in the IgE's, characterized in that it comprises a suspension of specific IgE-carrying basophil granulocytes, obtained from a blood sample taken from a human or animal patient who is being examined for parasitosis or allergy, which suspension contains from 300 to 100 basophils per mm.sup.3, by centrifugation to collect a layer containing essentially leucocytes, which is mixed with a buffer non-destructive relative to basophil cells, such as Hepes buffer or Pipes buffer for example, said homogenized mixture being then deposited on a liquid of density 1.079-1.085 whose osmolarity is comprised between 280 and 320 milliosmoles, to give rise, for centrifugation after a suitable period and at a suitable speed, to a ring which is constituted by a suspension enriched in basophils and which overlies the density liquid, which ring is taken off, then washed, preferably by means of the same buffer as above, to be deposited in different wells of a diagnosis read-out slide or dish.
Accordingly, also the present invention provides a method for the preparation of said diagnosis reagent, which comprises subjecting a blood sample taken from a human or animal patient assumed afflicted with a parasitosis or with an allergy, to a centrifugation for a suitable period and at a suitable speed to collect a leucocyte layer and a supernatant plasma rich in platelets; discarding said plasma and mixing the leucocyte layer with a suitable amount of buffer non-destructive with respect to basophil cells such as Hepes CM buffer or Pipes buffer, for example; injecting under said mixture a liquid of density 1.079-1.085, whose osmolarity is comprised between 280 and 320 milliosmoles, advantageously consisting of a mixture of sodium metrizoate and of Ficoll (Trademark registered by SIGMA) or of Percol (Trademark registered by PHARMACIA); centrifuging for a suitable period and at a suitable speed, to obtain a ring containing the basophils and the lymphocytes; and taking off said ring and washing it carefully with the same buffer as above which is discarded so as to preserve only the cellular cull, which, after resuspending, is ready to be deposited in the wells of a diagnosis read-out slide or dish.
According to an advantageous embodiment of the method of preparing the diagnosis reagent according to the present invention, the Hepes buffer non-destructive with respect to the basophil cells, by means of which the leucocyte layer separated by centrifugation is taken up again, has the following composition:
Hepes buffer, molar solution: 25 ml PA0 KCl 10%: 0.932 ml PA0 CaCl.sub.2 10%: 0.550 ml PA0 MgCl.sub.2 10%: 0.510 ml PA0 NaCl 9% (7.6 g): 844.11 ml PA0 H.sub.2 O q.s.p.: 1000 ml PA0 Hepes buffer, molar solution: 4 to 25 ml PA0 KCl 10%: 0.932 ml PA0 CaCl.sub.2 10%: 0.550 ml PA0 MgCl.sub.2 10%: 0.510 ml PA0 NaCl 9%: 850 to 900 ml PA0 Water q.s.p.: 1000 ml PA0 pH: 7.4-7.6 PA0 Hepes buffer, molar solution: 4 to 25 ml PA0 KCl 10%: 0.932 ml PA0 CaCl.sub.2 10%: 0.550 ml PA0 MgCl.sub.2 10%: 0.510 ml PA0 NaCl 9%: 850 to 900 ml PA0 Heparin: 10 to 15 units/ml PA0 Water q.s.p.: 1000 ml PA0 pH: 7.4-7.6 PA0 Hepes buffer, molar solution: 4 to 25 ml PA0 10% KCL: 0.932 to 1.2 ml PA0 9% NaCl: 850 to 900 ml PA0 Water q.s.p.: 1000 ml PA0 pH: 7.4-7.6 PA0 Toluidine blue or similar metachromatic dye: 2.2 g/liter approx. PA0 Aluminum sulfate: 7.5 g/liter approx. PA0 Cetylpyridinium halide: 0.75 g/liter approx. PA0 Glutaraldehyde: 20 g/liter approx. PA0 Formaldehyde: 35 g/liter approx. PA0 Absolute methanol: 300 ml/liter approx. PA0 Absolute ethanol: 300 ml/liter approx. PA0 Water q.s.p.: 1000 ml PA0 Toluidine blue or similar metachromatic dye: 0.2 to 0.8% PA0 Aluminum sulfate: 3 to 10% PA0 Cetylpyridinium halide: 0.3 to 0.8% PA0 Glutaraldehyde: 1.5 to 2.5% PA0 30% formaldehyde: 270 to 330 ml PA0 Methanol: 270 to 330 ml PA0 Ethanol: 270 to 330 ml PA0 Water q.s.p.: 1000 ml PA0 Toluidine blue or similar metachromatic dye: 0.2 to 0.8% PA0 Aluminum sulfate: 3 to 10% PA0 Cetylpyridinium halide: 0.3 to 0.8% PA0 30% formaldehyde: 300 ml .+-.10% PA0 Methanol: 300 ml .+-.10% PA0 Ethanol: 300 ml .+-.10% PA0 Water q.s.p.: 1000 ml PA0 Toluidine blue or similar metachromatic dye: 0.2 to 0.8% PA0 Aluminum sulfate: 3 to 10% PA0 Cetylpyridinium halide: 0.3 to 0.8% PA0 Methanol: 830 to 990 ml PA0 Water q.s.p.: 1000 ml PA0 Toluidine blue or similar metachromatic dye 0.2 to 0.8% PA0 Aluminum sulfate 3 to 10% PA0 Cetylpyridinium halide 0.3 to 0.8% PA0 Propyleneglycol 250 ml to 600 ml.+-.10% PA0 Methanol 200 ml to 750 ml.+-.10% PA0 Water q.s.p. 1000 ml PA0 Toluidine blue or similar metachromatic dye: 2.2 g/liter approx. PA0 Aluminum sulfate: 7.5 g/liter approx. PA0 Cetylpyridinium halide: 0.75 g/liter approx. PA0 Glutaraldehyde: 20 g/liter approx. PA0 Formaldehyde: 35 g/liter approx. PA0 Absolute methanol: 300 ml/liter approx. PA0 Absolute ethanol: 300 ml/liter approx. PA0 Water q.s.p.: 1000 ml PA0 Toluidine blue or similar metachromatic metachromatic dye: 0.2 to 0.8% PA0 Aluminum sulfate: 3 to 10% PA0 Cetylpyridinium halide: 0.3 to 0.3% PA0 Glutaraldehyde: 1.5 to 2.5% PA0 Formaldehyde 30%: 300 ml.+-.10% PA0 Methanol: 300 ml.+-.10% PA0 Ethanol: 300 ml.+-.10% PA0 Water q.s.p.: 1000 ml PA0 Toluidine blue or similar metachromatic dye: 0.2 to 0.8% PA0 Aluminum sulfate: 3 to 10% PA0 Cetylpyridinium halide: 0.3 to 0.8% PA0 30% Formaldehyde: 300 ml.+-.10% PA0 Methanol: 300 ml.+-.10% PA0 Ethanol: 300 ml.+-.10% PA0 Water q.s.p.: 1000 ml PA0 Toluidine blue or similar metachromatic dye: 0.2 to 0.8% PA0 Aluminum sulfate: 3 to 10% PA0 Cetylpyridinium halide: 1.3 to 1.8% PA0 Methanol: 380 to 990 ml PA0 Water q.s.p.: 1000 ml PA0 Toluidine blue or similar metachromatic dye: 0.2 to 0.8% PA0 Aluminum sulfate: 3 to 10% PA0 Cetylpyridinium halide: 0.3 to 0.8% PA0 Propyleneglycol: 250 ml to 600 ml.+-.10% PA0 Methanol: 200 ml to 750 ml.+-.10% PA0 Water q.s.p.: 1000 ml PA0 Hepes buffer, molar solution: 4 to 25 ml PA0 KCl to 10%: 0.932 ml PA0 CaCl.sub.2 10%: 0.550 ml PA0 MgCl.sub.2 10%: 0.510 ml PA0 NaCl 9%: 850 to 900 ml PA0 Heparin: 10 to 15 units/ml PA0 Water q.s.p.: 1000 ml PA0 pH: 7.4-7.6 PA0 Hepes buffer, molar solution: 4 to 25 ml PA0 KCl 10%: 0.932 to 1.2 ml PA0 NaCl 9%: 850 to 200 ml PA0 Water q.s.p.: 1000 ml
According to an advantageous embodiment of the method of preparing the diagnosis reagent according to the invention, the Hepes buffer which is non-destructive with respect to basophil cells, by means of which the leucocyte layer separated by centrifugation is taken up again, has a considerably reduced content of Hepes buffer, and a content of NaCl which is increased with respect to the contents of the above-defined buffer in these substances.
According to an advantageous feature of this embodiment, the quantitative composition of said Hepes buffer is preferably the following:
According to yet another embodiment of the method of preparing the diagnosis reagent according to the present invention, the sampling of a blood specimen in a human or animal patient assumed afflicted with a parasitosis or an allergy, is carried out on ethylenediamine-tetraacetic acid, in the form of one of its salts, and preferably in the form of its potassium salt (K.sub.3 EDTA), after which the total blood thus drawn, which can be preserved in this form for 24 hours, is mixed with an equal volume of a Hepes buffer of suitable composition, containing heparin in the proportion of 5 to 20 units/ml, then there may either be injected beneath this mixture a liquid of density 1.079-1.085 whose osmolarity is comprised between 280 and 320 milliosmoles, consisting in a mixture of sodium metrizoate and of "Ficoll" (Trademark registered by SIGMA), or of "Percol" (Trademark registered by PHARMACIA), or the heparinated blood Hepes buffer mixture may be transferred into a tube filled previously with the above-mentioned liquid of density 1.079-1.085, to separate the basophils, after which it is centrifuged for a suitable period and at a suitable speed, to obtain a ring containing the basophils and the lymphocytes, which is taken off and washed carefully with the above-said heparinated Hepes buffer which is discarded, after centrifugation, to preserve only the cellular culot which, after re-suspending, is ready to be deposited in the wells of a diagnosis read-out slide or dish.
Due to this modification of the method according to the invention, it is possible to treat blood taken the day before, with quite satisfactory results; in addition, the first centrifugation which, in the method of preparing the diagnosis reagent defined in the first place, follows immediately the taking of the blood specimen, is unnecessary, enabling a reduction in the time of the diagnosis test to at least one hour.
According to the present invention, the heparinated Hepes buffer applied, preferably, in this modification of the method of preparing the diagnosis reagent, advantageously has the following composition:
or the following composition:
in the latter case, the calcium and the magnesium in the form of their suitable salts, are placed in suitable amounts on the diagnosis read-out slides or dishes.
It is also an object of the present invention to provide a diagnosis method for parasitoses and allergies, and notably for worm parasitoses, which cause an increase in the circulating specific IgEs, characterized in that: the diagnosis reagent according to the present invention is placed in contact for about 15 minutes at 37.degree. C., with the specific antigen of the parasitosis or of the allergy that it is desired to diagnose, contained at different concentrations in a certain number of wells of a diagnosis read-out slide or dish, whilst the remaining wells of said slide or dish contain the diagnosis reagent alone, to serve as controls; there follows a rapid drying of the slide soprepared; it is followed by the simultaneous rapid fixing and dyeing, of the slide or of the dish thus prepared, by means of a simultaneous, rapid, hydro-alcoholic fixing and dyeing solution, of suitable composition, which contains a metachromatic dye which selectively dyes the cytoplasmic granules of the basophils, with which the slide or dish is put into contact for a short time, of the order of 5 to 15 minutes, for example; the basophil granulocytes are counted with the optical microscope, with a suitable enlargement (250 to 400 for example), respectively in the control wells and in the various wells containing the reagent and various antigen concentrations; and there is established, by comparison of the reduction in the number of basophil granulocytes (BG) in the wells containing the antigen at various concentrations, with respect to the number of BG contained in the control wells, the degranulation index ##EQU1## or percentage of basophils which have apparently disappeared in the presence of the antigen, which, if it is greater than 35%, enables the detection of the presence of the parasitosis or of the allergy sought.
According to an advantageous embodiment of the method of diagnosis according to the present invention, the simultaneous, rapid hydroalcoholic fixing and dyeing solution for the slides, is a solution with an acid pH, less than 2.5, which contains as the metachromatic dye, toluidine blue, associated with aluminum sulfate, a cetylpyridinium halide, glutaraldehyde and formaldehyde, in solution in a mixture of absolute ethanol and methanol and water.
According to an advantageous embodiment of the simultaneous, rapid fixing and dyeing reagent, applied in the method of diagnosis according to the invention, said reagent is constituted by a hydroalcoholic solution with an acid pH, less than 2.5, which has the following composition:
According to another advantageous embodiment of the simultaneous, rapid fixing and dyeing reagent solutions, according to the present invention, these solutions have the following quantitative composition:
According to yet another embodiment of these simultaneous, rapid fixing and dyeing solutions, the latter have the following qualitative and quantitative composition:
According to another embodiment of these simultaneous, rapid fixing and dyeing solutions, the latter have the following qualitative and quantitative composition:
According to another embodiment of the simultaneous, rapid fixing and dyeing solutions, the latter are distinguished by the fact that they are devoid of formaldehyde, of glutaraldehyde and of ethanol, and that they contain as fixing agent propylene-glycol.
According to an advantageous feature of this embodiment, the qualitative and quantitative composition of these solutions is as follows:
According to the present invention, the cetylpyridinium halide utilized is cetylpyridinium chloride or bromide.
In another aspect of the present invention it provides a ready-for-use set, or kit, preferably contained in a single case or box, for the performance of the diagnosis test for parasitoses and allergies, according to the present invention, which kit is characterized in that it comprises, in combination: a plurality of slides, dishes or the like each having a certain number of wells of equal diameter whose totality, except the wells which have to constitute controls, contain a pre-determined amount of a specific antigen of a parasitosis or of an allergy to be diagnosed, which antigen is introduced in the dry state, preferably lyophilized, into each of the wells, said pre-determined amounts varying from one column of wells to the following one; a plurality of tubes containing pre-measured amounts of a suitable buffer such as Herpes CM or Pipes buffer; a plurality of adapted containers such as ampoules, tubes, syringes or the like, each containing a liquid of density 1.079-1.085; at least one container containing a simultaneous rapid hydroalcoholic fixing and dyeing solution, of the above slides, dishes or the like, prepared according to the invention, whose pH is less than 2.5 and which contain a metachromatic dye such as preferably toluidine blue, aluminum sulfate, cetylpyridinium halide, glutaraldehyde and formaldehyde, in solution in a mixture of absolute methanol and ethanol and water.
According to an advantageous embodiment of the ready-for-use kit according to the present invention: each of the wells containing a specific antigen of the parasitosis or of the allergy to be diagnosed contains 10 to 20 .mu.l of said antigen in the lyophylized state; each tube containing a pre-measured amount of buffer containing 5 ml of said buffer; each ampoule, tube, syringe or the like, containing the density liquid contains 5 ml of said liquid of density 1.079-1.085; the or each container containing the simultaneous, rapid hydroalcoholic fixing and dyeing reagent for the prepared slides, dishes or similar, includes a solution of pH less than 2.5, having the following composition:
According to the invention, the slides, dishes or the like for diagnosis readout utilized, are selected notably from among: slides having a plurality of wells of equal diameter, constituted by paraffin rings; slides on which is glued an adhesive tape previously pierced by a plurality of holes of equal diameter which constitute the wells; transparent plastic dishes at the bottom of which is glued an adhesive tape pierced by a plurality of holes as indicated above.
According to an advantageous embodiment of the kit or set ready for use for the performance of the diagnosis test according to the present invention, the Hepes buffer contained in pre-measured amounts in ampoules or suitable tubes comprises from 4 to 25 ml of a molar solution of Hepes buffer, 0.932 ml of 10% KCl, 0.550 ml of 10% CaCl.sub.2, 0.510 ml of 10% MgCl.sub.2, from 850 to 900 ml of 9% NaCl, in solution in a sufficient amount of water to bring the solution to 1000 ml, and/or the simultaneous, rapid fixing and dyeing solution of the antigen preparations (or controls) borne by the diagnosis readout slides, contained in the corresponding container, has the following composition:
or the following composition:
or indeed the following composition:
or again the following composition:
According to an advantageous embodiment of the ready-for-use kit according to the present invention, for the performance of the diagnosis test, the latter is composed of a case which comprises: a plurality of adapted containers such as ampoules or tubes containing premeasured amounts of a suitable Hepes buffer, and notably Hepes buffer as defined above, a plurality of ampoules, of tubes or syringes, each containing premeasured amounts of a liquid of density 1.079-1.085, a plurality of diagnosis read-out slides or dishes each having a certain number of wells, of which the major part, with the exception of the control wells, are designed to receive variable predetermined amounts of an antigen specific to an allergy or parasitosis to diagnosed, and a plurality of ampoules or tubes, including premeasured amounts of simultaneous, rapid fixing and dyeing solution, of the above-defined type of solutions.
According to another advantageous embodiment of the ready-for-use kit according to the present invention, the latter is composed of two separate cases of which one, which is intended for the separation of the basophil cells, comprises: a plurality of adapted containers, such as tubes or ampoules containing premeasured amounts of a suitable buffer, notably of the Hepes buffer such as defined above, a plurality of ampoules or of tubes each containing a premeasured amount of a liquid of density 1.079-1.085; and of which the other case comprises: a plurality of diagnosis read-out slides or dishes each having a certain number of wells, of which the major part with the exception of the control wells, are intended to receive variable predetermined amounts of an antigen specific to an allergy or to a parasitosis to be diagnosed, and a plurality of ampoules or tubes including premeasured amounts of a simultaneous, rapid fixing and dyeing solution, or the type of solution defined above, for the performance of the diagnosis test proper.
According to yet another advantageous embodiment of the ready-for-use kit in one or two cases, according to the present invention, the Hepes buffer contained in premeasured amounts in a plurality of adapted containers, such as tubes or ampoules, has the following composition:
According to another advantageous embodiment of the ready-for-use kit in one or in two cases according to the present invention, the Hepes buffer contained in premeasured amounts in a plurality of adapted containers such as tubes or ampoules, has the following composition:
pH: 7.4-7.6
in which case, the diagnosis read-out slides or dishes include, in each of their wells designed to receive antigen, a predetermined amount of calcium and of magnesium in the form of their salts, and notably of their chlorides.
According to an advantageous feature of this embodiment, the calcium is present in the proportion of about 1 mmole/liter and the magnesium is present in the ratio of about 1 mmole/liter.
Besides the foregoing features, the invention comprises still other features, which will emerge from the description which follows.
The invention is aimed more particularly at novel diagnosis reagents, for parasitoses and allergies, as well as at their method of preparation, according to the foregoing features, and the diagnosis methods utilizing said reagent, as well as the means employed to produce said diagnosis methods.